REST-Activated Program of Gene Expression in Ischemia

 描述（由申请人提供）：REST，也称为NRSF，是一种基因沉默因子，在多能干细胞和神经祖细胞中广泛表达，在其中它积极地表达。 抑制大量对突触可塑性和结构重塑重要的编码和非编码基因，包括microRNA。REST和microRNA之间的相互作用是一个新兴的话题1 -4。新出现的证据表明microRNA在调节K+通道表达中起关键作用。我们的发现是，神经元损伤触发了成熟海马神经元中REST的诱导以及miR-132和Kv7.1表达（神经元功能所必需的靶基因）的REST依赖性沉默/减弱，这表明REST参与了缺血性卒中的发病机制11。我们的发现是，海马神经元中miR-132的过表达提供了强有力的保护，这证明了miR-132丢失与神经元死亡之间的因果关系，并强调了我们工作的临床相关性11。总体目标是研究我们最近在神经元中发现的两个经验证的REST靶点miR 132和KCNQ 1/Kv7.1在与全脑缺血相关的神经变性和认知障碍中的潜在作用。推动这项研究的中心假设是，全脑缺血上调了成年神经元中的REST，REST促进了一部分“转录响应”和基因网络的沉默，这些网络维持了神经元死亡和存活之间的平衡。我们试图在以下具体目的中检验这一假设：1）检查miR-132（一种候选REST靶标）在缺血诱导的神经元死亡中的作用。实验将1）验证miR-132响应于缺血性损伤而表现出REST依赖性表观遗传重塑和改变的基因表达; 2）确定REST是否与缺血诱导的miR-132表达和/或功能的改变有因果关系，所述缺血诱导的miR-132表达和/或功能的改变通过REST的过表达或抑制而在损伤的CA 1神经元中，包括floxed REST KO小鼠; 3）确定miR-132是否与缺血诱导的神经元死亡有因果关系; 4）通过miR pull-down测定鉴定促进神经元死亡的新的miR-132靶标; 5）检查缺血预处理防止缺血诱导的REST激活的能力，2）确定电压门控K+通道的Kv 7/KCNQ家族（新型REST靶基因）是否与缺血诱导的神经元死亡有因果关系。实验将：1）在控制和缺血条件下记录功能性KCNQ 1通道（候选REST靶标）的存在; 2）通过ChIP证实缺血对改变的REST结合和Kv7.1-3启动子处的表观遗传修饰的影响; 3）通过qPCR证实Kv7.1-3基因表达的改变，通过Westerns证实蛋白质和通过电生理学证实功能; 4）检查REST的遗传操作通过遗传操作模拟或预防缺血诱导的Kv7.1-3表达和/或功能改变的能力，包括REST cKO小鼠;和6）检查在神经元和REST的假定靶点中表达的新型microRNA（例如133和375）调节受损神经元中KCNQ 1表达和功能的能力。

 DESCRIPTION (provided by applicant): REST, also known as NRSF, is a gene silencing factor that is widely expressed in pluripotent stem cells and neural progenitors, where it actively represses a large array of coding and non-coding genes important to synaptic plasticity and structural remodeling including microRNAs. The interplay between REST and microRNAs is a new, emerging topic1-4. Emerging evidence indicates that microRNAs play a key role in regulation of K+ channel expression. Our findings that neuronal insults trigger induction of REST in mature hippocampal neurons and REST- dependent silencing/attenuation of miR-132 and Kv7.1 expression, target genes essential for neuronal function, implicate REST in the pathogenesis of ischemic stroke11. Our finding that overexpression of miR-132 in hippocampal neurons affords robust protection documents a causal relation between loss of miR-132 and neuronal death and underscores the clinical relevance of our work11. The overall objective is to examine a potential role for two validated REST targets miR132 and KCNQ1/Kv7.1, which we recently discovered in neurons, in the neurodegeneration and cognitive impairment associated with global ischemia. The central hypothesis driving the research is that global ischemia upregulates REST in adult neurons and that REST promotes silencing of a subset of "transcriptionally-responsive" and gene networks that maintain the balance between neuronal death and survival. We seek to test this hypothesis in the following Specific Aims: 1) Examine a role for miR-132, a candidate REST target, in ischemia-induced neuronal death. Experiments will 1) verify that miR-132 exhibits REST-dependent epigenetic remodeling and altered gene expression in response to ischemic insults; 2) Determine whether REST is causally related to ischemia-induced alterations in miR-132 expression and/or function in insulted CA1 neurons by overexpression or inhibition of REST, including a floxed REST KO mouse; 3) Determine whether miR-132 is causally related to ischemia-induced neuronal death; 4) Identify novel miR-132 targets that promote neuronal death by miR pull-down assay; 5) Examine the ability of ischemic preconditioning to prevent ischemia-induced activation of REST, suppression of miR-132 and rescue of neuronal death and cognitive deficits 2) Determine whether the Kv7/KCNQ family of voltage-gated K+ channels, novel REST target genes, are causally related to ischemia-induced neuronal death. Experiments will: 1) Document the presence of functional KCNQ1 channels, a candidate REST target, in under control and ischemic conditions; 2) Validate the impact of ischemia on altered REST binding and epigenetic modifications at Kv7.1-3 promoters by ChIP; 3) Validate alterations in gene expression of Kv7.1-3 by qPCR, protein by Westerns and function by electrophysiology; 4) Examine the examine ability of genetic manipulation of REST to mimic or prevent ischemia-induced alterations in Kv7.1-3 expression and/or function by genetic manipulation, including a REST cKO mouse; 5) Examine the ability of genetic manipulation of Kv7.1 to influence neuronal death and cognitive deficits; and 6) Examine the ability of novel microRNAs, such as 133 and 375, expressed in neurons and putative targets of REST, to regulate KCNQ1 expression and function in insulted neurons.